Immunodeficient rodents has revolutionized the study of regenerative medicine and cancer, to date, no analogous large animals with T, B, and NK-cell deficiency are available. We've used our novel multiplex gene- editing platform to knockout each allele of two genes, 1) Recombination Activating Gene (RAG2) and 2) Interleukin 2 Receptor, Gamma (IL2Rg) to establish RG-KO cell lines. This will dramatically reducing the time and cost associated with breeding individual genetic mutations into pigs. In addition, we propose to restore the immune systems in RG-KO pig by blastocyst complementation and cord blood transplantation. The restored immune function in these RG-KO swine will enable breeding animals to be maintained in standard housing with little risk of illness or death due to infection. In addition, breeding between chimeric, immune restored RG-KO's has nearly a 10-fold advantage over intercross of between heterozygous animals. This innovative method for establishment and breeding of immunodeficient provides a unique, sustainable supply of animals for research in regenerative medicine, xenogenic organ/tissue production and cancer using a large animal which is anatomically and physiologically similar to humans. This project will also establish a platform for the creation of a pig with a human immune system that would have profound impacts on the development and testing of novel therapeutics, transplantation/rejection studies, and vaccine development.